Philipp Henneke

Cutting edge: Immune stimulation by neisserial porins is toll-like receptor 2 and MyD88 dependent.

The immunopotentiating activity of neisserial porins, the major outer membrane protein of the pathogenic Neisseria, is mediated by its ability to stimulate B cells and up-regulate the surface expression of B7-2. This ability is dependent on MyD88 and Toll-like receptor (TLR)2 expression, as demonstrated by a lack of a response by B cells from MyD88 or TLR2 knockout mice to the porins. Using previously described TLR2-dependent reporter constructs, these results were confirmed and were shown to be due to induction of NF-κB nuclear translocation. This is the first demonstration of known vaccine adjuvant to stimulate immune cells via TLR2.

Toll-like receptor (TLR) signaling in response to Aspergillus fumigatus

Aspergillus fumigatus causes life-threatening infections in patients with qualitative and quantitative defects in phagocytic function. Here, we examined the contribution of Toll-like receptor (TLR)-2, TLR4, the adapter protein MyD88, and CD14 to signaling in response to the three forms of A. fumigatus encountered during human disease: resting conidia (RC), swollen conidia (SC), and hyphae (H). Compared with elicited peritoneal macrophages obtained from wild-type and heterozygous mice, TLR2−/− and MyD88−/− macrophages produced significantly less tumor necrosis factor-α (TNFα) followingA. fumigatus stimulation. In contrast, following stimulation with RC, SC, and H, TLR4−/− and CD14−/− macrophages exhibited no defects in tumor necrosis factor-α release. TLR2−/−, TLR4−/−, MyD88−/−, and CD14−/− macrophages bound similar numbers of RC and SC compared with wild-type macrophages. RC, SC, and H stimulated greater activation of a nuclear factor κ B (NFκB)-dependent reporter gene and greater release of tumor necrosis factor-α from the human monocytic THP-1 cell line stably transfected with CD14 compared with control cells stably transfected with empty vector. A. fumigatus stimulated NFκB-dependent reporter gene activity in the human embryonic kidney cell line, HEK293, only if the cells were transfected with TLR2. Moreover, activity increased when TLR2 and CD14 were co-transfected. Taken together, these data suggest that optimal signaling responses to A. fumigatus require TLR2 in both mouse and human cells. In contrast, a role for CD14 was found only in the human cells. MyD88 acts as a central adapter protein mediating signaling responses following stimulation with RC, SC, and H.

Induction of Tolerance to Lipopolysaccharide and Mycobacterial Components in Chinese Hamster Ovary/CD14 Cells Is Not Affected by Overexpression of Toll-Like Receptors 2 or 4

Down-regulation of cell surface expression of Toll-like receptor (TLR) 4 following LPS stimulation has been suggested to underlie endotoxin tolerance. In this study, we examined whether overexpression of TLR2 or TLR4 would affect the ability of cells to become tolerant to LPS or the mycobacterial components, arabinose-capped lipoarabinomannan (LAM) and soluble tuberculosis factor (STF). To this end, Chinese hamster ovary/CD14 cells stably transfected with a NF-κB-dependent reporter construct, endothelial leukocyte adhesion molecule CD25 (the 3E10 clone), were engineered to overexpress either human TLR2 or TLR4. Transfected TLRs exhibited proper signaling functions, as evidenced by increased LPS responsiveness of 3E10/TLR4 cells and acquisition of sensitivity to TLR2-specific ligands upon transfection of TLR2 into TLR2-negative 3E10 cells. Pretreatment of cells with LPS, LAM, or STF did not modulate TLR2 or TLR4 cell surface expression. Following LPS exposure, 3E10, 3E10/TLR2, and 3E10/TLR4 cells exhibited comparable decreases in LPS-mediated NF-κB activation and mitogen-activated protein (MAP) kinase phosphorylation. Likewise, LPS pretreatment profoundly inhibited LPS-induced NF-κB translocation in Chinese hamster ovary cells that concomitantly overexpressed human TLR4 and myeloid differentiation protein-2 (MD-2), but failed to modulate TLR4 or MD-2 cell surface expression. Pretreatment of 3E10/TLR2 cells with LAM or STF decreased their NF-κB responses induced by subsequent stimulation with these substances or LPS. Conversely, prior exposure of 3E10/TLR2 cells to LPS led to hyporesponsiveness to LPS, LAM, and STF, indicating that LPS and mycobacterial products induce cross-tolerance. Thus, tolerance to LPS and mycobacterial components cannot be attributed solely to a decrease in TLR/MD-2 expression levels, suggesting inhibition of expression or function of other signaling intermediates.

Novel Engagement of CD14 and Multiple Toll-Like Receptors by Group B Streptococci

Group B streptococcus (GBS) imposes a major health threat to newborn infants. Little is known about the molecular basis of GBS-induced sepsis. Both heat-inactivated whole GBS bacteria and a heat-labile soluble factor released by GBS during growth (GBS-F) induce nuclear translocation of NF-κB, the secretion of TNF-α, and the formation of NO in mouse macrophages. Macrophages from mice with a targeted disruption of MyD88 failed to secrete TNF-α in response to both heat-inactivated whole bacteria and GBS-F, suggesting that Toll-like receptors (TLRs) are involved in different aspects of GBS recognition. Immune cell activation by whole bacteria differed profoundly from that by secreted GBS-F. Whole GBS activated macrophages independently of TLR2 and TLR6, whereas a response to the secreted GBS-F was not observed in macrophages from TLR2-deficient animals. In addition to TLR2, TLR6 and CD14 expression were essential for GBS-F responses, whereas TLR1 and TLR4 or MD-2 did not appear to be involved. Heat lability distinguished GBS-F from peptidoglycan and lipoproteins. GBS mutants deficient in capsular polysaccharide or β-hemolysin had GBS-F activity comparable to that of wild-type streptococci. We suggest that CD14 and TLR2 and TLR6 function as coreceptors for secreted microbial products derived from GBS and that cell wall components of GBS are recognized by TLRs distinct from TLR1, 2, 4, or 6.

A Mechanism for Neurodegeneration Induced by Group B Streptococci through Activation of the TLR2/MyD88 Pathway in Microglia

Group B Streptococcus (GBS) is a major cause of bacterial meningitis and neurological morbidity in newborn infants. The cellular and molecular mechanisms by which this common organism causes CNS injury are unknown. We show that both heat-inactivated whole GBS and a secreted proteinaceous factor from GBS (GBS-F) induce neuronal apoptosis via the activation of murine microglia through a TLR2-dependent and MyD88-dependent pathway in vitro. Microglia, astrocytes, and oligodendrocytes, but not neurons, express TLR2. GBS as well as GBS-F induce the synthesis of NO in microglia derived from wild-type but not TLR2−/− or MyD88−/− mice. Neuronal death in neuronal cultures complemented with wild-type microglia is NO-dependent. We show for the first time a TLR-mediated mechanism of neuronal injury induced by a clinically relevant bacterium. This study demonstrates a causal molecular relationship between infection with GBS, activation of the innate immune system in the CNS through TLR2, and neurodegeneration. We suggest that this process contributes substantially to the serious morbidity associated with neonatal GBS meningitis and may provide a potential therapeutic target.

Cellular Activation, Phagocytosis, and Bactericidal Activity Against Group B Streptococcus Involve Parallel Myeloid Differentiation Factor 88-Dependent and Independent Signaling Pathways

Group B streptococci (GBS) vigorously activate inflammatory responses. We reported previously that a secreted GBS “factor” activates phagocytes via Toll-like receptor (TLR)2 and TLR6, but that GBS cell walls activate cells independently of these receptors. We hypothesized that the phagocytic immune functions in response to GBS, such as inflammation, uptake, and elimination of bacteria, occur through a coordinated engagement of TLRs, along with the coreceptors CD14 and CD11b/CD18. Using various knockout mice we show that GBS-induced activation of p38 and NF-κB depends upon the expression of the cytoplasmic TLR adapter protein, myeloid differentiation factor 88 (MyD88), but not TLR2 and/or TLR4. Macrophages with deletions of CD14 and complement receptor 3 had a normal cytokine response to whole bacteria, although the response to GBS factor was abrogated in CD14-null cells. The intracellular formation of bactericidal oxygen species proved to be MyD88 dependent; however, uptake of GBS, a prerequisite for intracellular killing by O2 radicals, occurred independently of MyD88. While deletion of complement receptor 3 greatly diminished the uptake of opsonized GBS, it did not affect the formation of bactericidal O2 radicals or inflammatory signaling intermediates. We conclude that the inflammatory, bactericidal, and phagocytic responses to GBS occur via parallel but independent processes.

Dual Role of TLR2 and Myeloid Differentiation Factor 88 in a Mouse Model of Invasive Group B Streptococcal Disease

Toll-like receptors (TLRs) are involved in pathogen recognition by the innate immune system. Different TLRs and the adaptor molecule myeloid differentiation factor 88 (MyD88) were previously shown to mediate in vitro cell activation induced by group B streptococcus (GBS). The present study examined the potential in vivo roles of TLR2 and MyD88 during infection with GBS. When pups were infected locally with a low bacterial dose, none of the TLR2- or MyD88-deficient mice, but all of the wild-type ones, were able to prevent systemic spread of GBS from the initial focus. Bacterial burden was higher in MyD88- than in TLR2-deficient mice, indicating a more profound defect of host defense in the former animals. In contrast, a high bacterial dose induced high level bacteremia in both mutant and wild-type mice. Under these conditions, however, TLR2 or MyD88 deficiency significantly protected mice from lethality, concomitantly with decreased circulating levels of TNF-α and IL-6. Administration of anti-TNF-α Abs to wild-type mice could mimic the effects of TLR2 or MyD88 deficiency and was detrimental in the low dose model, but protective in the high dose model. In conclusion, these data highlight a dual role of TLR2 and MyD88 in the host defense against GBS sepsis and strongly suggest TNF-α as the molecular mediator of bacterial clearance and septic shock.

c-Jun Kinase Is a Critical Signaling Molecule in a Neonatal Model of Group B Streptococcal Sepsis

Group B streptococcus (GBS) is the major cause of sepsis in newborn infants. In vitro, inactivated GBS stimulates macrophages to produce inflammatory proteins via the TLR adapter protein MyD88. Furthermore, inflammatory cytokine release in response to GBS greatly exceeds that following stimulation with pneumococci. In this study, we attempted to unravel signaling events that are involved in GBS-, but not Streptococcus pneumoniae-stimulated phagocytes to identify molecular targets for adjunctive sepsis therapy. We found that inactivated GBS and S. pneumoniae differed in the activation of the MAPK JNK, but not IκB kinase. Furthermore, JNK was essential for the transcriptional activation of inflammatory cytokine genes in response to GBS. Inhibition of JNK by the anthrapyrazolone SP600125 abrogated GBS-induced cytokine formation via an AP-1- and NF-κB-dependent mechanism without impairing antibacterial properties such as phagocytosis of GBS and the formation of intracellular oxidative species. In contrast, inhibition of the MAPK p38 impaired both antibacterial processes. In a neonatal mouse model of GBS sepsis SP600125 inhibited the inflammatory response and improved survival. In conclusion, JNK plays a major role in the inflammatory, but not in the direct antibacterial response to inactivated GBS, and may thus serve as a rational target for an adjunctive GBS sepsis therapy.

TIRAP: how Toll receptors fraternize.

Although some cellular responses induced by TLRs are abolished in MyD88-deficient mice, TLR4, unlike TLR9, can still induce activation of NF-κB and MAPKs. The discovery of a cytoplasmic adapter protein for TLR4, called TIRAP, helps explain this phenomenon.